Modification of both natural and synthetic polymers is an important method of creating new polymeric species with useful properties. For naturally occurring polymers, such modifications offer the possibility of producing polymers with the desired functional properties, while avoiding the expense and potential environmental costs associated with polymer synthesis based on petrochemical starting materials. Thus, for example, the most abundant natural polymer, cellulose, is widely used in modified forms, such as the carboxyalkylated and hydroxyalkylated derivatives.
While cellulose and its derivatives enjoy widespread usage, other natural and abundant polysaccharide polymers have been relatively under-exploited. One polymer derived from natural sources and offering potentially useful properties is chitosan. Chitosan is obtained by N-deacetylation of chitin, (C8H13NO5)n, a glucosamine polysaccharide that is structurally similar to cellulose. Chitin is a principal component of the shells of crustaceans, and is also found in other natural sources such as some insects, fungi, algae and yeast. Deacetylation of chitin by well-known methods such as those described in U.S. Pat. Nos. 4,282,351, 4,368,322 and 4,835,265, yields chitosan (Ia).

Chitosan is a linear polymer of glucosamine units. Structurally, it is distinguished from cellulose by the presence of the primary amine group. Chitosan is available commercially in various grades and average molecular weights (e.g., Sigma®, Aldrich®).
The presence of amine groups in chitosan confers interesting and potentially useful chemical and physical properties on the polymer. Although chitosan is not water soluble under neutral or alkaline conditions, under mildly acidic conditions (pH less than about 6) the amine groups are protonated and the polycationic polymer becomes water soluble (Ib). At neutral and alkaline pH, the amine groups are deprotonated and the neutral chitosan polymer is water-insoluble (Ia). The protonation-deprotonation, and accompanying change in water solubility, are reversible processes.

The failure to fully exploit these natural polymers lies, in part, on a lack of clean, effective and versatile processes to modify the polymers to provide needed functional properties. One such functional property is water solubility. Water-soluble polymers are becoming increasingly important compounds useful in a broad range of applications. Their importance lies, in part, in their ability to function in environmentally “friendly” ways. Only relatively few polymers, however, whether natural or synthetic, are water-soluble. The most abundant natural polymers, for example, the polysaccharides cellulose and chitin, are linear polymers with poor water solubility. Although chitosan has substantial solubility in aqueous solution, it is soluble only at low pH. In neutral and basic aqueous solutions, chitosan is essentially insoluble.
Several approaches are possible to solubilize water-insoluble natural polymers. One approach is simply to reduce the polymer's average molecular weight by breaking up polymer chains into smaller chains. Such a molecular weight reduction approach is often impractical, as the resultant polymer may lose desirable physical or biological properties.
A more practical approach is to chemically modify natural polymers to add enough hydrophilic or charged side groups to confer on the polymer the desired degree of water solubility. For example, whereas natural cellulose is insoluble in water, its carboxylated derivatives such as carboxymethylcellulose are an important class of water-soluble natural polymers. Unfortunately, polymer modification reactions typically employ reagents whose use presents environmental problems. A common scheme for the carboxymethylation of cellulose, for example, uses the chlorinated reagent chloroacetic acid, a highly toxic and corrosive chlorinated compound. The potential environmental and safety problems are further exacerbated when the polymeric starting material is itself a synthetic polymer rather than a natural polymer, since such polymers are typically produced using organic solvents and petrochemical-based monomers, the widespread use of which creates additional environmental concerns.
Several schemes for the chemical modification of chitosan have been reported. Carboxymethylation of chitosan can be achieved by treatment with chloroacetic acid (hydroxy nucleophilic reaction), as is done with cellulose, or by reduction of a chitosan glyoxylate (amino nucleophilic reaction) with sodium cyanoborohydride. Muzzarelli et al., “N-(carboxymethylidene)chitosans and N-(carboxymethyl)chitosans: Novel Chelating Polyampholytes Obtained from Chitosan Glyoxylate,” Carbohydrate Research, 107, 199-214 (1982). The N-(carboxymethyl)chitosan products reportedly are soluble in aqueous solutions over a range of pH values. Similarly, soluble carbohydrate derivatives of chitosan have been reported by a reductive alkylation process, again using sodium cyanoborohydride. Yalpani et al., “Some Chemical and Analytical Aspects of Polysaccharide Modifications. 3. Formation of Branched-Chain, Soluble Chitosan Derivatives”, Macromolecules, 17, 272-281 (1984). Other soluble chitosan derivatives are known. For example, U.S. Pat. No. 5,378,472 discloses a 5-methylpyrrolidinone chitosan that is reportedly soluble in aqueous alkaline solution. Although these chemical modification approaches can yield chitosan derivatives with altered functional properties (e.g., base solubility), the reagents used to achieve these modifications (e.g., chloroacetic acid and sodium cyanoborohydride) have undesirable properties with respect to health and safety. Moreover, an effective, versatile method of modifying chitosan to provide other useful functional properties is also needed.
One promising approach to producing modified natural polymers is to make use of clean and selective enzymatic reactions. Such reactions could be used, for example, to add hydrophilic side groups or charged groups to polymers to confer enhanced water solubility. Other side groups could also be added in enzyme-catalyzed reactions to change the physical and chemical properties of the polymer. Enzyme modification potentially offers a number of advantages over conventional chemical modification. Enzyme reactions typically do not involve the use of highly reactive reagents, thus avoiding many potential health and safety hazards. In addition, enzyme reactions can be highly selective, and such selectivity can be exploited to eliminate the number of reaction steps necessary to produce the desired product, e.g., by eliminating the need for wasteful protection and deprotection steps.
Some groups have reported successful enzyme-based polymer modifications. In one approach, hydrolytic enzymes are used under non-aqueous conditions to catalyze reactions such as condensation or transesterification. Bruno et al. has reported the transesterification of a caprate group onto amylose, using a protease in an isooctane solvent. Bruno et al., “Enzymatic modification of insoluble amylose in organic solvents,” Macromolec., 28:8881-8883 (1995). This approach, however, is subject to severe steric limitations. The reaction mechanism involves formation of an acyl-enzyme intermediate, which undergoes nucleophilic attack at the enzyme's active site, requiring both the acyl intermediate and the polymeric substrate to bind to the enzyme active site. Because of these steric limitations, such reactions, while selective, are quite slow.
In another approach, the above-mentioned steric limitations are eliminated by using a two-step reaction. In a first reaction, the enzyme and substrate react to generate a reactive intermediate species. The intermediate, non-enzyme-bound species diffuses freely through the reaction solvent and reacts remotely with the polymer surface. This approach has been used to graft phenols onto lignin polymers, using a peroxidase. Popp et al., “Incorporation of p-cresol into lignins via peroxidase-catalyzed copolymerization in nonaqueous media,” Enzyme Microb. Technol., 13:964-968 (1991); Blinkovsky et al., “Peroxidase-catalyzed synthesis of lignin-phenol copolymers,” J. Polym. Sci., 31:1839-1846 (1993). Similarly, this approach has been used to enzymatically create epoxide groups in polybutadiene, using a lipase with hydrogen peroxide to generate a reactive peroxycarboxylic acid intermediate. Jarvie et al., “Enzymatic epoxidation of polybutadiene,” Chem. Comm., 177-178 (1998).
Other work has focused on the use of chitosan films or gels in enzyme-catalyzed heterogeneous phase water treatment applications. For example, U.S. Pat. No. 5,340,483 discloses a method of selectively removing phenolic components in wastewater mixtures by reacting the phenolic compound with the enzyme tyrosinase in the presence of chitosan. Enzymes such as tyrosinases, phenol oxidases and polyphenol oxidases are known to react with a wide variety of phenolic compounds. Without wishing to be bound by theory, it is believed that these enzymes act to reduce the phenol to a reactive ortho quinone, as shown in (II).
Quinones in turn react with the chitosan film or gel to form a tightly-bound chemisorbed species, thereby effectively removing the phenol from solution by an enzyme-catalyzed chitosan reaction. Such reaction schemes have important applicability to wastewater treatment and waste management.
Efforts to exploit tyrosinase-catalyzed reactions to create chitosan derivatives with important functional properties have been reported. Payne et al., “Enzyme-based polymer modification: Reaction of phenolic compounds with chitosan films,” Polymer, 37, 4643-4648 (1996). These modification approaches employ insoluble chitosan films, and this approach suffers because only the surface of the chitosan gel or film is exposed to the quinone intermediate in this heterogeneous phase process. Other heterogeneous-phase chitosan-tyrosinase reaction schemes are described, for example, in Lenhart et al., “Enzymatic Modification of Chitosan by Tyrosinase,” in Enzymes in Polymer Synthesis (Gross et al., eds.), American Chemical Society, pp. 188-198 (1998), the disclosure of which is incorporated herein by reference in its entirety.
Although there have been some successes in enzyme-modification of polymers, these biotechnological approaches suffer from a number of practical disadvantages. Many such reactions are too slow to be commercially practical, are not readily controlled, employ prohibitively expensive reagents, or are limited by the surface-orientated nature of heterogeneous phase reactions as described above.
It is thus desirable to develop new chitosan derivatives which have important functional properties such as broad water-solubility. It is further desirable to develop new ways to produce such polymers without employing hazardous chemicals or generating potentially environmentally hazardous waste streams. It is particularly desirable to develop ways to modify naturally-occurring polymers to confer desired functional properties in simple, versatile, and environmentally clean ways.